Template:Kyoto/Notebook/Sep 5

From 2013.igem.org

(Difference between revisions)
(→Electrophoresis)
(→Restriction Enzyme Digestion)
 
(44 intermediate revisions not shown)
Line 1: Line 1:
==Sep 5==
==Sep 5==
-
===Colony PCR===
 
-
<div class="experiment">
 
-
<span class="author">No name</span>
 
-
{| class="wikitable"
 
-
!Sample||base pair
 
-
|-
 
-
|8/9 J23100-3||1142
 
-
|-
 
-
|8/9 J23100-4||1142
 
-
|-
 
-
|8/9 J23100-5||1142
 
-
|}
 
-
{| class="wikitable"
 
-
!PreDenature||Denature||Annealing||Extension||cycle
 
-
|-
 
-
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
 
-
|-
 
-
|5m||30s||30s||30s||30cycles
 
-
|}
 
-
</div>
 
-
 
===Electrophoresis===
===Electrophoresis===
<div class="experiment">
<div class="experiment">
Line 37: Line 16:
|5||100bp ladder||--||--
|5||100bp ladder||--||--
|}
|}
-
[[File:igku_xx.xxx]]<br>
+
[[File:igku_Sep5_electrophoresis_N1.jpg]]<br>
</div>
</div>
Line 75: Line 54:
|}
|}
incubate 16 &deg;C 1 hour  
incubate 16 &deg;C 1 hour  
 +
</div>
 +
 +
===Electrophoresis===
 +
<div class="experiment">
 +
<span class="author">Tatsui</span>
 +
{| class="wikitable"
 +
!Lane||Sample
 +
|-
 +
|1||100bp ladder
 +
|-
 +
|2||RBS-luxI-DT
 +
|-
 +
|3||Pbad/araC
 +
|-
 +
|4||NC
 +
|-
 +
|5||100bp ladder
 +
|}
 +
[[File:igku_Sep5_electrophorsis_N2_1.jpg]]<br>
 +
[[File:igku_Sep5_electrophorsis_N2_2.jpg]]<br>
</div>
</div>
Line 89: Line 88:
|3||RBS-luxI-DT||
|3||RBS-luxI-DT||
|-
|-
-
|4||1kbp ladder||
+
|4||||
|-
|-
|5||Pbad/araC||
|5||Pbad/araC||
Line 95: Line 94:
|6||Pbad/araC||
|6||Pbad/araC||
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:Igku Sep5 Gel Extraction(N3) 1.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:Igku Sep5 Gel Extraction(N3) 2.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 111: Line 110:
!Sample||base pair
!Sample||base pair
|-
|-
-
|9/4 apt12_1R(pSB1C3)-1||384
+
|9/4 aptamer 12_1R(pSB1C3)-1||384
|-
|-
-
|9/4 apt12_1R(pSB1C3)-2||384
+
|9/4 aptamer 12_1R(pSB1C3)-2||384
|-
|-
|9/4 pT181 antisense(pSB1C3)-1||415
|9/4 pT181 antisense(pSB1C3)-1||415
Line 134: Line 133:
!Sample||base pair
!Sample||base pair
|-
|-
-
|9/4 apt12_1R-DT-1||521
+
|9/4 aptamer 12_1R-DT-1||521
|-
|-
-
|9/4 apt12_1R-DT-2||521
+
|9/4 aptamer 12_1R-DT-2||521
|-
|-
-
|9/4 apt12_1R-DT-3||521
+
|9/4 aptamer 12_1R-DT-3||521
|-
|-
-
|9/4 apt12_1R-DT-4||521
+
|9/4 aptamer 12_1R-DT-4||521
|-
|-
|9/4 pT181 attenuator(pSB1C3)||601
|9/4 pT181 attenuator(pSB1C3)||601
Line 181: Line 180:
|5min||30s||30s||72s||30cycles
|5min||30s||30s||72s||30cycles
|}
|}
-
</div>
 
-
 
-
===Electrophoresis===
 
-
<div class="experiment">
 
-
<span class="author">No name</span>
 
-
{| class="wikitable"
 
-
!Lane||Sample||Enzyme1||Enzyme2
 
-
|-
 
-
|1||100bp ladder||--||--
 
-
|-
 
-
|2||apt12_1R (pSB1C3)-1||--||--
 
-
|-
 
-
|3||apt12_1R (pSB1C3)-2||--||--
 
-
|-
 
-
|4||pT181 antisense(pSB1C3)-1||--||--
 
-
|-
 
-
|5||pT181 antisense(pSB1C3)-2||--||--
 
-
|-
 
-
|6||pT181 antisense(pSB1C3)-3||--||--
 
-
|-
 
-
|7||pT181 antisense(pSB1C3)-4||--||--
 
-
|-
 
-
|8||100bp ladder||--||--
 
-
|}
 
-
[[File:igku_xx.xxx]]<br>
 
-
</div>
 
-
{| class="wikitable"
 
-
!Lane||Sample||Enzyme1||Enzyme2
 
-
|-
 
-
|1||100bp ladder||--||--
 
-
|-
 
-
|2||apt12_1R-DT-1||--||--
 
-
|-
 
-
|3||apt12_1R-DT-2||--||--
 
-
|-
 
-
|4||apt12_1R-DT-3||--||--
 
-
|-
 
-
|5||apt12_1R-DT-4||--||--
 
-
|-
 
-
|6||pT181 attenuator(pSB1C3)-1||--||--
 
-
|-
 
-
|7||pT181 attenuator(pSB1C3)-2||--||--
 
-
|-
 
-
|8||RBS-lysis1-DT-1||--||--
 
-
|-
 
-
|9||RBS-lysis1-DT-2||--||--
 
-
|-
 
-
|10||100bp ladder||--||--
 
-
|}
 
-
[[File:igku_xx.xxx]]<br>
 
-
</div>
 
-
{| class="wikitable"
 
-
!Lane||Sample||Enzyme1||Enzyme2
 
-
|-
 
-
|1||100bp ladder||--||--
 
-
|-
 
-
|2||RBS-lysis2-DT-1||--||--
 
-
|-
 
-
|3||RBS-lysis2-DT-2||--||--
 
-
|-
 
-
|4||RBS-lysis2-DT-3||--||--
 
-
|-
 
-
|5||RBS-lysis2-DT-4||--||--
 
-
|-
 
-
|6||RBS-lysis3-DT-1||--||--
 
-
|-
 
-
|7||RBS-lysis3-DT-2||--||--
 
-
|-
 
-
|8||100bp ladder||--||--
 
-
|}
 
-
[[File:igku_xx.xxx]]<br>
 
</div>
</div>
Line 281: Line 209:
|9/5 Ptet+pT181 antisense||2||20||22||--
|9/5 Ptet+pT181 antisense||2||20||22||--
|-
|-
-
|9/5 Plux+pT181 attenuater||2||20||22||--
+
|9/5 Plux+pT181 attenuator||2||20||22||--
|-
|-
-
|9/5 Ptet+pT181 attenuater||2||20||22||--
+
|9/5 Ptet+pT181 attenuator||2||20||22||--
|-
|-
-
|9/5 Ptet+pT181 attenuater||2||20||22||--
+
|9/5 Ptet+pT181 attenuator||2||20||22||--
|-
|-
|9/5 Pconst+RBS-tetR+DT||2||20||22||--
|9/5 Pconst+RBS-tetR+DT||2||20||22||--
Line 298: Line 226:
<span class="author">No name</span>
<span class="author">No name</span>
{| class="wikitable"
{| class="wikitable"
-
! ||9/5 Pbad/araC||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/5 Pbad/araC||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||4&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 18&micro;L|| 30&micro;L
|2 cuts||4&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L|| 18&micro;L|| 30&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/5 RBS-luxI-DT||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/5 RBS-luxI-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||4&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||18&micro;L|| 30&micro;L
|2 cuts||4&micro;L|| 0&micro;L|| 0&micro;L|| 1&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||18&micro;L|| 30&micro;L
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/28 Plux||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/28 Plux||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts|| 12&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||10&micro;L||30&micro;L
|2 cuts|| 12&micro;L|| 0&micro;L|| 1&micro;L|| 0&micro;L|| 1&micro;L|| 3&micro;L|| 3&micro;L||10&micro;L||30&micro;L
Line 315: Line 243:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/5 Pconst(J23100)||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/5 Pconst(J23100)||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||5.7&micro;L||0&micro;L||1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||16.3&micro;L||30&micro;L
|2 cuts||5.7&micro;L||0&micro;L||1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||16.3&micro;L||30&micro;L
Line 322: Line 250:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||9/5 Plac||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||9/5 Plac||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||6.0&micro;L||0&micro;L||1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||16&micro;L||30&micro;L
|2 cuts||6.0&micro;L||0&micro;L||1&micro;L||0&micro;L||1&micro;L||3&micro;L||3&micro;L||16&micro;L||30&micro;L
Line 329: Line 257:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/31 Plux-RBS-GFP-DT-1||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/31 Plux-RBS-GFP-DT-1||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||9.4&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||12.6&micro;L||30&micro;L
|2 cuts||9.4&micro;L||1&micro;L||1&micro;L||0&micro;L||0&micro;L||3&micro;L||3&micro;L||12.6&micro;L||30&micro;L
Line 336: Line 264:
|}
|}
{| class="wikitable"
{| class="wikitable"
-
! ||8/20 Pconst-RBS-luxR-DT||E||S||X||P||BSA||Buffer||MilliQ||total
+
! ||8/20 Pconst-RBS-luxR-DT||EcoRI||SpeI||XbaI||PstI||BSA||Buffer||MilliQ||total
|-
|-
|2 cuts||4.5&micro;L||1&micro;L||0&micro;L||1&micro;L||0&micro;L||3&micro;L||3&micro;L||13.5&micro;L||30&micro;L
|2 cuts||4.5&micro;L||1&micro;L||0&micro;L||1&micro;L||0&micro;L||3&micro;L||3&micro;L||13.5&micro;L||30&micro;L
Line 355: Line 283:
<div class="experiment">
<div class="experiment">
<span class="author">No name</span>
<span class="author">No name</span>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||aptamer 12_1R (pSB1C3)-1||--||--
 +
|-
 +
|3||aptamer 12_1R (pSB1C3)-2||--||--
 +
|-
 +
|4||pT181 antisense(pSB1C3)-1||--||--
 +
|-
 +
|5||pT181 antisense(pSB1C3)-2||--||--
 +
|-
 +
|6||pT181 antisense(pSB1C3)-3||--||--
 +
|-
 +
|7||pT181 antisense(pSB1C3)-4||--||--
 +
|-
 +
|8||100bp ladder||--||--
 +
|}
 +
[[File:Igku Sep5 Electrophoresis(N4).jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||aptamer 12_1R-DT-1||--||--
 +
|-
 +
|3||aptamer 12_1R-DT-2||--||--
 +
|-
 +
|4||aptamer 12_1R-DT-3||--||--
 +
|-
 +
|5||aptamer 12_1R-DT-4||--||--
 +
|-
 +
|6||pT181 attenuator(pSB1C3)-1||--||--
 +
|-
 +
|7||pT181 attenuator(pSB1C3)-2||--||--
 +
|-
 +
|8||RBS-lysis1-DT-1||--||--
 +
|-
 +
|9||RBS-lysis1-DT-2||--||--
 +
|-
 +
|10||100bp ladder||--||--
 +
|}
 +
[[File:Igku Sep5 Electrophoresis(N6) 2.jpg]]<br>
 +
{| class="wikitable"
 +
!Lane||Sample||Enzyme1||Enzyme2
 +
|-
 +
|1||100bp ladder||--||--
 +
|-
 +
|2||RBS-lysis2-DT-1||--||--
 +
|-
 +
|3||RBS-lysis2-DT-2||--||--
 +
|-
 +
|4||RBS-lysis2-DT-3||--||--
 +
|-
 +
|5||RBS-lysis2-DT-4||--||--
 +
|-
 +
|6||RBS-lysis3-DT-1||--||--
 +
|-
 +
|7||RBS-lysis3-DT-2||--||--
 +
|-
 +
|8||100bp ladder||--||--
 +
|}
 +
[[File:Igku Sep5 Electrophoresis(N5).jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Lane||Sample||Enzyme1||Enzyme2
!Lane||Sample||Enzyme1||Enzyme2
Line 362: Line 354:
|2||Pconst||SpeI||PstI
|2||Pconst||SpeI||PstI
|-
|-
-
|3||Pconst||--||--
+
|3||Pconst NC||--||--
|-
|-
|4||Plac||SpeI||PstI
|4||Plac||SpeI||PstI
|-
|-
-
|5||Plac||--||--
+
|5||Plac NC||--||--
|-
|-
|6||Plux||SpeI||PstI
|6||Plux||SpeI||PstI
|-
|-
-
|7||Plux||--||--
+
|7||Plux NC||--||--
|-
|-
|8||Pconst-RBS-luxR-DT||EcoRI||XbaI
|8||Pconst-RBS-luxR-DT||EcoRI||XbaI
|-
|-
-
|9||Pconst-RBS-luxR-DT||--||--
+
|9||Pconst-RBS-luxR-DT NC||--||--
|-
|-
|10||Plux-RBS-GFP-DT||EcoRI||SpeI
|10||Plux-RBS-GFP-DT||EcoRI||SpeI
|-
|-
-
|11||Plux-RBS-GFP-DT||--||--
+
|11||Plux-RBS-GFP-DT NC||--||--
|-
|-
|12||1kbp ladder||--||--
|12||1kbp ladder||--||--
|}
|}
-
[[File:igku_xx.xxx]]<br>
+
[[File:Igku Sep5 Electrophoresis(N6) 3.jpg]]<br>
</div>
</div>
-
===5*M9(EDTA)===
+
===5x M9 Medium (+EDTA)===
<div class="experiment">
<div class="experiment">
<span class="author">Hirano</span>
<span class="author">Hirano</span>
Line 399: Line 391:
|NH4Cl||10mg
|NH4Cl||10mg
|-
|-
-
|Fe(3)-EDTA||1263.27g　
+
|Fe(III)-EDTA||1263.27mg　
 +
|-
 +
|MilliQ||up to 10 mL
|}
|}
 +
*autoclave at 121 &deg;C for 20 min
</div>
</div>
-
121&deg;C 20minute autoclave
 
===Gel Extraction===
===Gel Extraction===
Line 416: Line 410:
|3||Pconst||SpeI & PstI
|3||Pconst||SpeI & PstI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_Sep5_gel_extraction_N7_1.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_Sep5_gel_extraction_N7_2.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 436: Line 430:
|6||Plux||SpeI & PstI
|6||Plux||SpeI & PstI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_Sep5_gel_extraction_N7_3.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_Sep5_gel_extraction_N7_4.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 458: Line 452:
|6||Plux-RBS-GFP-DT||EcoRI & SpeI
|6||Plux-RBS-GFP-DT||EcoRI & SpeI
|}
|}
-
[[File:igku_xxbeforexx.xxx]]<br>
+
[[File:igku_Sep5_gel_extraction_N7_5.jpg]]<br>
-
[[File:igku_xxafterxx.xxx]]<br>
+
[[File:igku_Sep5_gel_extraction_N7_6.jpg]]<br>
{| class="wikitable"
{| class="wikitable"
!Name||concentration[&micro;g/mL]||260/280||260/230
!Name||concentration[&micro;g/mL]||260/280||260/230
Line 467: Line 461:
|Plux-RBS-GFP-DT (EcoRI & SpeI)||21.8||1.87||0.98
|Plux-RBS-GFP-DT (EcoRI & SpeI)||21.8||1.87||0.98
|}
|}
 +
 +
===DNA Purification===
 +
<div class="experiment">
 +
<span class="author">No name</span>
 +
{|class="wikitable"
 +
!DNA||concentration[&micro;g/mL]||260/280||260/230
 +
|-
 +
|9/5 RBS-luxI-DT||7.6||1.56||-1.42
 +
|-
 +
|9/5 Pbad/araC||7.5||1.54||3.84
 +
|}
 +
</div>
===Miniprep===
===Miniprep===
Line 500: Line 506:
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
|94&deg;C||94&deg;C||55&deg;C||68&deg;C||--
|-
|-
-
|5m||30s||30s||1m 25s||30cycles
+
|5min||30s||30s||1min 25s||30cycles
|}
|}
</div>
</div>
-
===Electrophoresis===
+
===Liquid Culture===
<div class="experiment">
<div class="experiment">
-
<span class="author">No name</span>
+
<span class="author">Hirano</span>
{| class="wikitable"
{| class="wikitable"
-
!Lane||Sample||Enzyme1||Enzyme2
+
!Sample||medium
|-
|-
-
|1||100bp ladder||--||--
+
|9/4 aptamer 12_1R-DT-1||Plusgrow medium (+CP)
|-
|-
-
|2||RBS-lysis3-DT-3||--||--
+
|9/4 pT181 attenuator(pSB1C3)-1||Plusgrow medium (+CP)
|-
|-
-
|3||RBS-lysis3-DT-4||--||--
+
|9/4 RBS-lysis2-DT-1||Plusgrow medium (+CP)
-
|-
+
-
|4||RBS-lysis3-DT-5||--||--
+
-
|-
+
-
|5||RBS-lysis1-DT-3||--||--
+
-
|-
+
-
|6||Ptrc KaiC-1||--||--
+
-
|-
+
-
|7||pSB4K5||--||--
+
-
|-
+
-
|8||100bp ladder||--||--
+
-
|}
+
-
</div>
+
-
 
+
-
===Liquid Culture===
+
-
<div class="experiment">
+
-
<span class="author">Hirano</span>
+
-
{| class="wikitable"
+
-
!Sample||medium
+
|-
|-
-
|JM109-1(Master plate)||LB medium
+
|9/4 Ptrc-KaiC -1||Plusgrow medium (+Amp)
|-
|-
 +
|9/4 pSB4K5 -1||Plusgrow medium (+Kan)
|}
|}
</div>
</div>

Latest revision as of 14:20, 27 September 2013

Contents

Sep 5

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2J23100-3----
3J23100-4----
4J23100-5----
5100bp ladder----

Igku Sep5 electrophoresis N1.jpg

Miniprep

Nakamoto

DNAconcentration[µg/mL]260/280260/230
8/3 J23100-3349.11.691.851
8/10 Plac-2331.31.841.42

Ligation

Kojima

stateVectorInserterLigation High ver.2
experimentPconst13.2spinach-DT2.13.5
experimentPlac15.3spinach-DT2.13.5
experimentPconst13.2pT181 antisense (XbaI & PstI)6.63.5
experimentPlac15.3pT181 antisense (XbaI & PstI)6.63.5
experimentPtet30.1pT181 antisense (XbaI & PstI)6.63.5
experimentPconst13.2pT181 attenuator11.73.5
experimentPlac15.3pT181 attenuator11.73.5
experimentPtet30.1RBS-lacZα-DT3.03.5

incubate 16 °C 1 hour

Electrophoresis

Tatsui

LaneSample
1100bp ladder
2RBS-luxI-DT
3Pbad/araC
4NC
5100bp ladder

Igku Sep5 electrophorsis N2 1.jpg
Igku Sep5 electrophorsis N2 2.jpg

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder
2RBS-luxI-DT
3RBS-luxI-DT
4
5Pbad/araC
6Pbad/araC

Igku Sep5 Gel Extraction(N3) 1.jpg
Igku Sep5 Gel Extraction(N3) 2.jpg

Nameconcentration[µg/mL]260/280260/230
RBS-luxI-DT23.20.837.89
Pbad/araC51.81.200.11

Colony PCR

No name

Samplebase pair
9/4 aptamer 12_1R(pSB1C3)-1384
9/4 aptamer 12_1R(pSB1C3)-2384
9/4 pT181 antisense(pSB1C3)-1415
9/4 pT181 antisense(pSB1C3)-2415
9/4 pT181 antisense(pSB1C3)-3415
9/4 pT181 antisense(pSB1C3)-4415
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s30s30cycles
Samplebase pair
9/4 aptamer 12_1R-DT-1521
9/4 aptamer 12_1R-DT-2521
9/4 aptamer 12_1R-DT-3521
9/4 aptamer 12_1R-DT-4521
9/4 pT181 attenuator(pSB1C3)601
9/4 pT181 attenuator(pSB1C3)601
9/4 RBS-lysis1-DT613
9/4 RBS-lysis1-DT613
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s36s30cycles
Samplebase pair
9/4 RBS-lysis2-DT-1985
9/4 RBS-lysis2-DT-2985
9/4 RBS-lysis2-DT-3985
9/4 RBS-lysis2-DT-4985
9/4 RBS-lysis3-DT-11210
9/4 RBS-lysis3-DT-21210
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s72s30cycles

Liquid Culture

Nakamoto

Samplemedium
8/9 J23100-5Plusgrow medium(+Amp)

37°C

Transformation

No name

NameSample(µL)Competent Cells(µL)Total(µL)Plate
9/5 Pconst+spinach-DT22022--
9/5 Plac+spinach-DT22022--
9/5 Pconst+pT181 antisense22022--
9/5 Plac+pT181 antisense22022--
9/5 Ptet+pT181 antisense22022--
9/5 Plux+pT181 attenuator22022--
9/5 Ptet+pT181 attenuator22022--
9/5 Ptet+pT181 attenuator22022--
9/5 Pconst+RBS-tetR+DT22022--
9/16(2012) T7-His-FT22022--
9/16(2012) pBr32222022--

Restriction Enzyme Digestion

No name

9/5 Pbad/araCEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4µL 0µL 1µL 0µL 1µL 3µL 3µL 18µL 30µL
9/5 RBS-luxI-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4µL 0µL 0µL 1µL 1µL 3µL 3µL18µL 30µL
8/28 PluxEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts 12µL 0µL 1µL 0µL 1µL 3µL 3µL10µL30µL
NC 0.6µL0µL0µL 0µL 0µL1µL1µL7.4µL10µL
9/5 Pconst(J23100)EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts5.7µL0µL1µL0µL1µL3µL3µL16.3µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
9/5 PlacEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts6.0µL0µL1µL0µL1µL3µL3µL16µL30µL
NC 0.3µL0µL0µL 0µL 0µL1µL1µL7.7µL10µL
8/31 Plux-RBS-GFP-DT-1EcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts9.4µL1µL1µL0µL0µL3µL3µL12.6µL30µL
NC 0.5µL0µL0µL 0µL 0µL1µL1µL7.5µL10µL
8/20 Pconst-RBS-luxR-DTEcoRISpeIXbaIPstIBSABufferMilliQtotal
2 cuts4.5µL1µL0µL1µL0µL3µL3µL13.5µL30µL
NC 0.2µL0µL0µL 0µL 0µL1µL1µL7.8µL10µL
DNAconcentration[µg/mL]260/280260/230
9/5 RBS-luxI-DT7.61.56-1.42
9/5 Pbad/araC7.51.543.84

Electrophoresis

No name

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2aptamer 12_1R (pSB1C3)-1----
3aptamer 12_1R (pSB1C3)-2----
4pT181 antisense(pSB1C3)-1----
5pT181 antisense(pSB1C3)-2----
6pT181 antisense(pSB1C3)-3----
7pT181 antisense(pSB1C3)-4----
8100bp ladder----

Igku Sep5 Electrophoresis(N4).jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2aptamer 12_1R-DT-1----
3aptamer 12_1R-DT-2----
4aptamer 12_1R-DT-3----
5aptamer 12_1R-DT-4----
6pT181 attenuator(pSB1C3)-1----
7pT181 attenuator(pSB1C3)-2----
8RBS-lysis1-DT-1----
9RBS-lysis1-DT-2----
10100bp ladder----

Igku Sep5 Electrophoresis(N6) 2.jpg

LaneSampleEnzyme1Enzyme2
1100bp ladder----
2RBS-lysis2-DT-1----
3RBS-lysis2-DT-2----
4RBS-lysis2-DT-3----
5RBS-lysis2-DT-4----
6RBS-lysis3-DT-1----
7RBS-lysis3-DT-2----
8100bp ladder----

Igku Sep5 Electrophoresis(N5).jpg

LaneSampleEnzyme1Enzyme2
11kbp ladder----
2PconstSpeIPstI
3Pconst NC----
4PlacSpeIPstI
5Plac NC----
6PluxSpeIPstI
7Plux NC----
8Pconst-RBS-luxR-DTEcoRIXbaI
9Pconst-RBS-luxR-DT NC----
10Plux-RBS-GFP-DTEcoRISpeI
11Plux-RBS-GFP-DT NC----
121kbp ladder----

Igku Sep5 Electrophoresis(N6) 3.jpg

5x M9 Medium (+EDTA)

Hirano

volume10ml
Na2HPO4 60mg
KH2PO430mg
NaCl5mg
NH4Cl10mg
Fe(III)-EDTA1263.27mg　
MilliQup to 10 mL
  • autoclave at 121 °C for 20 min

Gel Extraction

No name

LaneDNAEnzyme
11kbp ladder
2PconstSpeI & PstI
3PconstSpeI & PstI

Igku Sep5 gel extraction N7 1.jpg
Igku Sep5 gel extraction N7 2.jpg

Nameconcentration[µg/mL]260/280260/230
Pconst5.31.980.06
LaneDNAEnzyme
1100bp ladder
2PlacSpeI & PstI
3PlacSpeI & PstI
5PluxSpeI & PstI
6PluxSpeI & PstI

Igku Sep5 gel extraction N7 3.jpg
Igku Sep5 gel extraction N7 4.jpg

Nameconcentration[µg/mL]260/280260/230
Plac (SpeI & PstI)5.21.820.36
Plux(SpeI & PstI)8.01.870.25
LaneDNAEnzyme
11kbp ladder
2Pconst-RBS-luxR-DTEcoRI & XbaI
3Pconst-RBS-luxR-DTEcoRI & XbaI
5Plux-RBS-GFP-DTEcoRI & SpeI
6Plux-RBS-GFP-DTEcoRI & SpeI

Igku Sep5 gel extraction N7 5.jpg
Igku Sep5 gel extraction N7 6.jpg

Nameconcentration[µg/mL]260/280260/230
Pconst-RBS-luxR-DT(EcoRI & XbaI)30.61.841.16
Plux-RBS-GFP-DT (EcoRI & SpeI)21.81.870.98

DNA Purification

No name

DNAconcentration[µg/mL]260/280260/230
9/5 RBS-luxI-DT7.61.56-1.42
9/5 Pbad/araC7.51.543.84

Miniprep

No name

DNAconcentration[µg/mL]260/280260/230
8/9 J23100-5243.01.901.81

Colony PCR

No name

Samplebase pair
9/4 RBS-lysis3-DT-31210
9/4 RBS-lysis3-DT-41210
9/4 RBS-lysis3-DT-51210
9/4 RBS-lysis1-DT-3613
9/4 Ptrc KaiC-1--
9/4 pSB4K51370
PreDenatureDenatureAnnealingExtensioncycle
94°C94°C55°C68°C--
5min30s30s1min 25s30cycles

Liquid Culture

Hirano

Samplemedium
9/4 aptamer 12_1R-DT-1Plusgrow medium (+CP)
9/4 pT181 attenuator(pSB1C3)-1Plusgrow medium (+CP)
9/4 RBS-lysis2-DT-1Plusgrow medium (+CP)
9/4 Ptrc-KaiC -1Plusgrow medium (+Amp)
9/4 pSB4K5 -1Plusgrow medium (+Kan)
Retrieved from "http://2013.igem.org/Template:Kyoto/Notebook/Sep_5"